PKC beta I mediates the inhibition of P2Y receptor-induced inositol phosphate formation in endothelial cells

1 Bovine pulmonary artery endothelium (CPAE) expresses phospholipase C (PLC )-linked P2Y(1) and P2Y(2) receptors, for them 2-methylthio-ATP (2MeSATP) a nd UTP are respective agonists. Here, we have investigated the particular p rotein kinase C (PKC) isoform(s) responsible for the inhibition of P2Y(1) a nd P2Y(2) receptor-evoked inositol phosphate (IP) formation by phorbol 12-m yristate 13-acetate (PMA). 2 Although short-term (20 min) pretreatment of cells with PMA attenuated 2M eSATP- and UTP-induced phosphoinositide (PI) breakdown, this inhibition was lost after 15 h. Preincubation with PMA for 24 h, on the contrary, potenti ated 2MeSATP and UTP responses. The TP formation stimulated by NaF was unal tered by PMA pretreatment. 3 Western blot analysis showed that treatment of CPAE with PMA resulted in a rapid translocation of PKC isoform beta I, epsilon and mu, but not lambda , from the cytosol to the membrane fraction. 4 Pretreatment of the selective PKC inhibitor Ro 31-8220 attenuated the inh ibitory effect of PMA on IP formation. Go 6976 (an inhibitor of conventiona l PKC alpha, beta and gamma) and LY 379196 (a selective PKC beta inhibitor) also dose-dependently inhibited the PMA-mediated desensitization. 5 Transfection of PKC beta-specific antisense oligonucleotide reduced PKC b eta I protein level and inhibited PMA-mediated PI reduction. 6 RT-PCR analysis showed that PMA treatment for 4-24 h up-regulated P2Y(1) and P2Y(2) receptors at the mRNA levels. 7 These results suggest that PKC beta I may exert a negative feedback regul ation on endothelial P2Y(1) and P2Y2 receptor-mediated PI turnover. The dow n-regulation of PKC beta I and enhanced P2Y receptor expression together mi ght contribute to the late PI enhancing effect of PMA.